The field of the invention is plant seed-associated promoters.
There is substantial interest in identification and isolation of regulatory elements that control gene expression in plant seed; such seed-specific and seed-associated promoters may be used in the generation of transgenic plants with desired seed traits. For instance, seed specific regulatory elements are useful for manipulating lipid metabolism, especially fatty acid synthesis in seed, and for enhancing agronomic traits such as herbicide and pesticide resistance and drought tolerance.
Seed storage proteins genes are among the most tightly regulated plant genes. Seed storage protein expression is highly seed-specific, and the corresponding transcripts accumulate to high levels in the middle to late stages of seed development. Many seed storage protein genes have been cloned from diverse plant species, and their promoters have been analyzed in detail (see, e.g., Thomas, 1993, Plant Cell 5:1401–1410). Promoter elements, which constitute the 5′-upstream regulatory regions, have been functionally defined by their ability to confer seed-specific expression of the bacterial beta-glucuronidase (GUS) reporter gene in transgenic plants (e.g., Bogue et al. 1990, Mol Gen Genet 222:49–57; Bustos et al. 1989, Plant Cell 1:839–853). Deletion analysis of these promoters has allowed researchers to define regions within each promoter that are critical to its overall regulation (Bustos et al. 1991, EMBO J. 10:1469–1479; Chung, 1995, Ph.D. Dissertation, Texas A&M University; Nunberg et al 1994, Plant Cell 6:473–486).
In some cases, cis-regulatory elements have been mapped and the trans-acting factors that confer functionality have been cloned. The cis-acting elements that regulate seed storage protein expression have been found in genes from a variety of plant species including rice, sunflower, French bean and soybean. Conserved nucleotide sequences that are commonly found in seed-specific regulatory regions have been identified and include the legumin-box (leg-box), which comprises a core element of CATGCATG, also called an RY repeat element.
The napin promoter (from the napA gene encoding the Brassica napus, 2S storage protein) is widely used to control expression of lipid biosynthetic genes in transgenic plants (Josefsson et al., 1987, J Biol Chem 262:12196–201; Stalberg et al., 1993, Plant Mol Biol 23:671–83; Ellerstrom et al., 1996, Plant Mol Biol 32:1019–27). The Brassica napus oleosin promoter directs reporter gene expression in the embryo and endosperm (Keddie et al., 1994, Plant Mol Biol 24:327–40). In Arabidopsis, the FAE1 promoter has been used to control expression of the GUS reporter in developing embryos, where activity was detected as early as 4–5 days after fertilization (Rossak et al., 2001, Plant Mol Biol 46:717–25). The legumin promoter (LeB4; Baumlein et al., 1991, Mol Gen Genet 225:121–8; Baumlein et al., 1992, Plant J 2:233–9) from the legume Vicia faba has also been functionally characterized and shown to promote seed-specific expression of heterologous genes. The characterization of seed-specific promoters was reviewed in Goossens et al., 1999 (Plant Pyhsiol 120:1095–1104).
The vast majority of native plant promoters are unidirectional, with one upstream (5′) promoter directing only one gene that is 3′ to the promoter. Bidirectional promoters exist in some prokaryotes and viruses, and bi-directional promoters that are active in plants have been identified from viral (geminivirus; Frey et al., 2001, Virus Genes 22:231–42) and bacterial (Agrobacterium; Schmulling et al., 1989, Plant Cell 1:665–70; Leung et al., 1991, Mol Gen Genet 230:463–74) plant pathogens. A strategy for engineering bidirectional promoters, essentially by adding a minimal promoter region to the upstream end of a uni-directional promoter, has been proposed (Xie et al., 2001, Nat Biotechnol 19:677–9). Additionally, a bidirectional promoter from Brassica napus has been characterized wherein the regulatory region controls seed-specific expression in one direction while in the opposite orientation, the promoter directs expression in a variety of tissues including leaves and roots (Keddie et al., 1994, Plant Mol Biol 1994 24:327–40; Sadanandom et al., 1996, Plant J 10:235–42). However, no plant promoters that control seed-associated expression in both orientations have been described.
Globulins and albumins are common seed storage proteins in dicotyledonous plants (dicots). These proteins can be distinguished from each other based on differential solubility; albumins are water soluble, whereas globulins are soluble in salt solutions. In many dicots, including citrus and almond (Prunus amygdalus), 12S globulins are the most prevalent seed storage proteins. 12S globulins are multimeric proteins composed of dimer subunits. Each dimer contains a 30–40 kDa alpha-subunit and a 20 kDa beta-subunit. The dimer subunits are derived from a single precursor polypeptide that undergoes several post-translational modifications, including cleavage of a signal peptide followed by cleavage of the pre-protein into the alpha and beta subunits.
Two full-length cDNA clones encoding the prunin 12S globulin seed storage proteins (Pru1 and Pru2) have been isolated from almond (Prunus amygdalus cv Texas; Garcia-Mas et al., 1995, Plant Mol Biol 27:205–10). Each transcript encodes a pre-protein that is processed to create the alpha and beta subunits that comprise the ˜60 kDa mature seed storage protein. At the amino acid level, Pru1 and Pru2 are 63% identical, with Pru2 containing two gaps in the region corresponding to the alpha subunit. The two prunin transcripts are seed-specific and are among the most abundant in immature seeds. It appears that the main storage proteins in almond are legumin-like globulins that consist of two main pairs of polypeptides, each pair encoded by a single gene (Garcia-Mas et al., 1995).